WO2023125566A1 - 具有抑菌功能的酵母蛋白及其制备方法 - Google Patents

具有抑菌功能的酵母蛋白及其制备方法 Download PDF

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WO2023125566A1
WO2023125566A1 PCT/CN2022/142484 CN2022142484W WO2023125566A1 WO 2023125566 A1 WO2023125566 A1 WO 2023125566A1 CN 2022142484 W CN2022142484 W CN 2022142484W WO 2023125566 A1 WO2023125566 A1 WO 2023125566A1
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yeast
enzymolysis
milk
protein
temperature
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French (fr)
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覃先武
徐智鹏
胡骏鹏
戴晋军
谢智文
黄鑫
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Angel Yeast Co Ltd
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Angel Yeast Co Ltd
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Priority to EP22914818.4A priority patent/EP4458978A4/en
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01006—Endo-1,3(4)-beta-glucanase (3.2.1.6)
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    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • the application relates to the field of yeast deep processing, in particular, to a yeast protein with antibacterial function and a preparation method thereof.
  • yeast hydrolyzate and yeast cell wall were introduced into the catalog of feed ingredients, they have been more and more recognized in the industry, and there are more and more uses for yeast hydrolyzate and yeast cell wall.
  • the use of yeast hydrolyzate-like yeast protein raw materials can not only alleviate the shortage of feed protein resources, but also improve the nutritional quality of protein raw materials.
  • the use of yeast cell walls to improve immunity and promote the health of animals has been gradually accepted by people, and it is an ideal substitute for antibiotics in feed.
  • yeast hydrolyzate production process it can provide nutrition and growth-promoting function in the field of cultivation, but it has no antibacterial effect.
  • yeast protein products with antibacterial functions develop yeast protein products with antibacterial functions to achieve inhibition of Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus) and other bacteria, and achieve broad-spectrum Antibacterial effect, so as to realize the upgrading of the product function of the yeast protein series products, has far-reaching significance.
  • the yeast protein with antibacterial function can not only provide nutrition to promote growth, but also prevent bacterial diseases, promote growth while strengthening immunity, and help fully reduce the impact of the ban on antibiotics in breeding.
  • the main purpose of this application is to provide a yeast protein with antibacterial function and its preparation method, to solve the problem that the existing yeast hydrolyzate and yeast cell wall products do not have antibacterial function, and it is difficult to prevent bacterial diseases in the process of breeding without antibiotics technical problem.
  • a method for preparing a yeast protein with antibacterial function includes the following steps: step S1, performing liquid fermentation on the Saccharomyces cerevisiae strain, and then separating to obtain yeast milk; wherein The Saccharomyces cerevisiae strain is selected from Saccharomyces cerevisiae FX-2 CCTCC NO:M2016418 and/or Saccharomyces cerevisiaed8.8 CCTCC NO:M2017148; step S2, acidifying the yeast milk to obtain acidified yeast milk; optionally, the yeast Carrying out autolysis of the milk, or exogenous enzyme-catalyzed hydrolysis, or mechanical crushing, to obtain a cell wall emulsion; step S3, mixing acidified yeast milk and an optional cell wall emulsion and performing autolysis to obtain an autolysis mixture; wherein the autolysis mixture
  • the concentration of dry yeast is 10-20% based on the mass content of dry yeast; step S4, using compound protease to
  • a carbon source, a nitrogen source and a phosphorus source are added in a fed-batch manner during the liquid fermentation process, the carbon source is molasses, the nitrogen source is ammonia water, and the phosphorus source is ammonium dihydrogen phosphate solution; preferably, the molasses
  • the total sugar content is 28.5-31.5%
  • the nitrogen element content in the ammonia water is 15-17%
  • the phosphorus element content in the ammonium dihydrogen phosphate solution is 9-11%.
  • the temperature in the liquid fermentation process is 30-35 °C
  • the fermentation time is 12-16 hours
  • the fermentation pH value is 4.5-6.0
  • the protein content in the yeast milk is ⁇ 50%.
  • step S1 includes: performing liquid fermentation on Saccharomyces cerevisiaed8.8 CCTCC NO:M2017148, and then separating to obtain the first yeast milk; performing liquid fermentation on Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418, and then separating to obtain the second Yeast milk; in step S2, the first yeast milk is acidified to obtain acidified yeast milk; the second yeast milk is autolyzed, or exogenous enzyme catalyzed hydrolysis, or mechanically crushed to obtain a cell wall emulsion; in step S3, the The acidified yeast milk and the cell wall emulsion are mixed and autolyzed to obtain an autolyzed mixture.
  • step S2 citric acid is used to acidify the first yeast milk; preferably, the amount of citric acid added is 1-4% of the weight of yeast milk; preferably, in step S2, the protein content in the cell wall emulsion is 15% to 30%; preferably, step S3 includes: mixing acidified yeast milk and cell wall emulsion to obtain a premix with a protein content ⁇ 35%; adjusting the solid content of the premix and then using steam to heat up to autolyze to obtain an autolyzed mixture liquid; more preferably, during the process of raising the temperature of the steam for autolysis, the temperature is controlled at 45-55° C., and the holding time is 8-14 hours.
  • the composite protease includes papain, neutral protease, alkaline protease, acid protease; preferably, the composite protease is protease EF108.
  • the amount of compound protease added is 4-8 ⁇ of the dry basis weight in the autolyzed mixture; preferably, the enzymolysis temperature of the first enzymolysis process is 55-60°C, The pH is 5.0-6.0, and the enzymatic hydrolysis time is 6-12 hours.
  • the amount of mannase added is 4-8 ⁇ of the dry basis weight in the first enzymolysis liquid; preferably, the enzymolysis temperature of the second enzymolysis process is 55-60 °C, pH 5.0-6.0, enzymolysis time 6-12h.
  • the addition amount of ⁇ -glucanase is 3 ⁇ 6 ⁇ of the dry basis weight in the second enzymolysis solution, and the addition amount of cellulase is the dry weight in the second enzymolysis solution.
  • the enzymolysis temperature in the third enzymolysis process is 45-55°C, the pH is 4.0-5.0, and the enzymolysis time is 8-16h; more preferably, the third enzymatic hydrolysis In the hydrolysis process, use organic acid and/or inorganic acid to adjust the pH value to 4.0 ⁇ 5.0; More preferably, organic acid is one or more in malic acid, citric acid, lactic acid, and inorganic acid is hydrochloric acid and/or Sulfuric acid, more preferably an organic acid is used for conditioning.
  • step S6 includes: raising the temperature of the yeast protein solution to 78-82°C to kill enzymes for 1-2 hours; evaporating and concentrating the yeast protein solution after the enzyme-killing until the solid content reaches 35-45%, to obtain a bacteriostatic Functional yeast protein; preferably, after the evaporative concentration step, the preparation method further includes: spray-drying the concentrate obtained in the evaporative concentration step to obtain yeast protein with antibacterial function; more preferably, during the spray-drying process, further The air temperature is controlled at 155-175°C, the outlet air temperature is controlled at 90-115°C, the atomization pressure is controlled at 100-160bar, and the receiving temperature is controlled at 25-45°C.
  • a yeast protein with antibacterial function is also provided, which is prepared by the above preparation method.
  • This application uses a specific strain of Saccharomyces cerevisiae to obtain yeast milk raw materials and optional yeast cell wall raw materials. After mixing the acidified yeast milk and optional yeast cell wall emulsion, a specific enzymatic hydrolysis process is used to enzymolyze the mixture. After evaporation and concentration, a yeast protein product with antibacterial function is formed. It increases the antibacterial function on the basis of meeting the functional indicators of yeast protein products such as protein (protein ⁇ 35%) and small peptides, and develops a product with a wide range of antibacterial properties. Yeast protein products with spectrum antibacterial function. The yeast protein can prevent bacterial diseases during the antibiotic-free breeding process and play a role in strengthening immunity.
  • Saccharomyces cerevisiae FX-2 was deposited in the China Center for Typical Microorganisms Collection (CCTCC) on August 1, 2016, with the preservation number CCTCCNO: M2016418, and the preservation address: China. Wuhan. Wuhan University, postal code: 430072; Tel ( 027)-68754052.
  • the brewing strain Saccharomyces cerevisiaed8.8 was preserved in the China Center for Typical Microorganisms Collection (CCTCC) on March 29, 2017, the preservation number is CCTCC NO: M2017148, the preservation address: China. Wuhan. Wuhan University, postal code: 430072; Tel ( 027)-68754052.
  • Fig. 1 shows the bacteriostasis experiment result of the yeast protein prepared according to the examples and comparative examples of the present application to Escherichia coli;
  • Fig. 2 shows the results of antibacterial experiments on Staphylococcus aureus by yeast proteins prepared according to Examples and Comparative Examples of the present application.
  • the existing yeast hydrolyzate and yeast cell wall products have no inhibitory effect on the growth of Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus). An effective zone of inhibition could not be formed in the zone test.
  • the application provides a method for preparing yeast protein with antibacterial function. This application realizes the functional upgrade of the yeast water protein product, and realizes the inhibition of the yeast protein of the mixed product on Gram-negative bacteria (E. Spectrum antibacterial function.
  • a method for preparing yeast protein with antibacterial function includes the following steps: Step S1, performing liquid fermentation on Saccharomyces cerevisiae strains, and then isolating yeast milk; wherein Saccharomyces cerevisiae The yeast strain is selected from Saccharomyces cerevisiae FX-2 CCTCC NO:M2016418 and/or Saccharomyces cerevisiaed8.8 CCTCC NO:M2017148; step S2, acidifying the yeast milk to obtain acidified yeast milk; optionally, the yeast milk Perform autolysis, or exogenous enzyme-catalyzed hydrolysis, or mechanical crushing to obtain a cell wall emulsion; step S3, mix acidified yeast milk and an optional cell wall emulsion and perform autolysis to obtain an autolysis mixture; wherein the autolysis mixture The concentration is 10-20% based on an appropriate amount of dry yeast; Step S4, using a compound protease to perform the first enzymolysis on the autolyze
  • This application uses a specific strain of S.
  • the enzymatic hydrolysis process enzymolyzes the mixed liquid, and then evaporates and concentrates to form a yeast protein product with antibacterial function.
  • the yeast protein product contains protein, nucleic acid, small peptide, dextran and mannan, etc., and its dissolution rate can reach 45-65%.
  • the antibacterial function is added, and a yeast protein product with broad-spectrum antibacterial function has been developed.
  • the yeast protein can replace antibiotics and be used in the field of livestock and poultry breeding, can prevent bacterial diseases, reduce the impact caused by the prohibition of antibiotics, and play a role in strengthening immunity.
  • a carbon source, a nitrogen source and a phosphorus source are added in a liquid fermentation process, and the carbon source is Molasses, the nitrogen source is ammonia, and the phosphorus source is ammonium dihydrogen phosphate solution (aqueous solution); preferably, the total sugar content in the molasses is 28.5 to 31.5% (mass content), and the content of nitrogen in ammonia is 15 to 17% ( mass content), the phosphorus element content in the ammonium dihydrogen phosphate solution is 9 to 11% (mass content); preferably, the temperature in the liquid fermentation process is 30 to 35°C, the fermentation time is 12 to 16h, and the fermentation pH is 4.5-6.0; preferably, the protein content in the yeast milk is ⁇ 50% (mass content).
  • the yeast milk obtained in step S1 can be divided into two parts, one part is used for acidification to form acidified yeast milk, and the other part is used for preparing cell wall emulsion, or different Saccharomyces cerevisiae strains can be used Different yeast milks were prepared to prepare acidified yeast milk and cell wall emulsion. Of course, it is also possible to use only acidified yeast milk for subsequent enzymatic hydrolysis.
  • step S1 includes: carrying out liquid fermentation of Saccharomyces cerevisiaed8.8 CCTCC NO:M2017148, and then separating and obtaining the first yeast milk; Saccharomyces cerevisiae FX-2 CCTCCNO: M2016418 is subjected to liquid fermentation, and then separated to obtain the second yeast milk; in step S2, acidifying the first yeast milk to obtain acidified yeast milk; autolyzing the second yeast milk, or exogenous enzyme catalysis Hydrolysis or mechanical crushing to obtain cell wall emulsion; in step S3, acidified yeast milk and the cell wall emulsion are mixed and autolyzed to obtain the autolyzed mixture.
  • step S2 the yeast milk is acidified with citric acid; preferably, the amount of citric acid added is 1 to 4% of the weight.
  • step S2 the protein content in the cell wall emulsion is 15-30%; preferably, step S3 includes: mixing the acidified yeast milk and the cell wall emulsion to obtain a premix solution with a protein content ⁇ 35% ( More preferably, 5-35% of the cell wall emulsion is added to the acidified yeast milk based on the proportion of solids; after adjusting the solid content of the premixed solution, the temperature of the premixed solution is increased by steam to perform autolysis to obtain an autolyzed mixed solution. Mixing the two according to the protein content ⁇ 35% is conducive to balancing the content of each component in the final product, and is also conducive to further improving the antibacterial effect of the yeast protein product.
  • the temperature is controlled at 45-55° C., and the holding time is 8-14 hours. More preferably, when adjusting the dry matter, the solid content in the premix is adjusted to 10-20%.
  • the composite protease includes papain, neutral protease, alkaline protease, and acid protease; preferably, the composite protease is protease EF108, a composite enzyme preparation of Angel Anzhanmei.
  • the above-mentioned composite protease is used to proteolyze the autolyzed mixture, and the macromolecules contained in the yeast cells are hydrolyzed into small molecular polypeptides by proteolysis, and the spatial structure is opened, exposing many specific and functional groups, which is more conducive to Improve the nutrient content and antibacterial properties of the product.
  • the amount of compound protease added is 4-8 ⁇ of the dry basis weight in the autolyzed mixture; preferably, The enzymolysis temperature of the first enzymolysis process is 55-60° C., the pH is 5.0-6.0, and the enzymolysis time is 6-12 hours.
  • the amount of mannase added is 4-8 ⁇ of the dry basis weight in the first enzymolysis solution;
  • the enzymolysis temperature of the second enzymolysis process is 55-60° C.
  • the pH is 5.0-6.0
  • the enzymolysis time is 6-12 hours.
  • the amount of ⁇ -glucanase added is 3-3% of the dry basis weight in the second enzymatic hydrolysis solution. 6 ⁇ , the amount of cellulase added is 1-3 ⁇ of the dry basis weight in the second enzymolysis solution; preferably, the enzymolysis temperature in the third enzymolysis process is 45-55°C, and the pH is 4.0-5.0 , Enzymolysis time is 8 ⁇ 16h.
  • organic acid and/or inorganic acid is used to adjust the pH value to 4.0-5.0; more preferably, the organic acid is one or more of malic acid, citric acid, and lactic acid , the inorganic acid is hydrochloric acid and/or sulfuric acid, more preferably an organic acid is used for adjustment.
  • step S6 includes: raising the temperature of the yeast protein solution to 78-82°C to inactivate the enzyme for 1-2 hours; evaporating and concentrating the yeast protein solution after the enzyme inactivation until the solid content reaches 35-45 %, the yeast protein with antibacterial function was obtained. Evaporating and concentrating to form a paste product also has the bacteriostatic effect described in this application. The specific evaporation and concentration process can be carried out by using a three-effect or four-effect evaporation system.
  • the preparation method further includes: spray-drying the concentrated solution obtained in the evaporating and concentrating step to obtain yeast protein with antibacterial function. It is easy to store and transport after spray drying. The temperature of spray drying will directly affect the moisture and color of the product. If the temperature is too low, the product will be lighter in color and have a high moisture content, making it difficult to control the product hygiene index. If the temperature is high, the product will be darker in color and less in moisture, making it difficult to control the product quality.
  • the inlet air temperature is controlled at 155-175°C
  • the outlet air temperature is controlled at 90-115°C
  • the atomization pressure is controlled at 100-160bar
  • the receiving temperature material packaging temperature
  • the yeast milk is autolyzed, hydrolyzed by exogenous enzymes, or mechanically broken to obtain a cell wall emulsion.
  • a yeast protein with antibacterial function is also provided, which is prepared by the above preparation method.
  • the raw materials of the yeast protein product with antibacterial function are all derived from natural yeast, and large-scale production can be realized.
  • the yeast protein with antibacterial function obtained in this application on the basis of maintaining crude protein and small peptide nutrients, increases the content of zymosan compared with single yeast hydrolyzate, and additionally improves immune function.
  • the polysaccharide hydrolyzate is additionally used to inhibit bacteria, forming a yeast protein with antibacterial function .
  • this application first fully uses protease to fully enzymolyze the proteins in yeast cells and cell walls, exposing many functional groups and functional components, and then uses polysaccharase to enzymolyze yeast cell walls, so that yeast cells And the antibacterial active substances in the yeast cell wall are fully released to form an antibacterial effect.
  • the yeast protein with antibacterial function developed by this application can alleviate a series of effects brought about by the prohibition of antibiotics in breeding, promote animal growth, improve the transformation rate, and improve the breeding level.
  • the application has passed small experimental test, production pilot test and large-scale production, and the production method is feasible.
  • the yeast strain is a strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae FX-2 CCTCCNO: M2016418, Saccharomyces cerevisiaed8.8, CCTCC NO: M2017148) culture solution contains carbon source, nitrogen source, phosphorus source, wherein the carbon source is 30%
  • the sugarcane molasses the nitrogen source is ammonia water with a nitrogen content of 16%
  • the phosphorus source is phosphoric acid.
  • Saccharomyces cerevisiae is inoculated for fermentation and culture.
  • the carbon source, nitrogen source, and phosphorus source are all added by fed-batch method.
  • the fermentation temperature is 30°C and the fermentation time is After 17 hours, the fermentation pH was controlled at 5.5, and the pure culture liquid fermented yeast milk was obtained, and the yeast milk protein was determined to be 46%.
  • the yeast milk prepared by the above process of Saccharomyces cerevisiaed8.8 was used to make acidified yeast milk: based on the mass of yeast milk dry matter, 4% citric acid was added to acidify the yeast milk to obtain acidified yeast milk.
  • yeast milk prepared by the above process of Saccharomyces cerevisiae FX-2 is used to make cell wall emulsion: yeast milk autolysis treatment, the yeast autolysis process controls the salt concentration to 6%, the pH value is 6.5, and the temperature is 55°C. After autolysis for 25 hours, centrifuge After separation, a yeast cell wall emulsion was obtained with a protein content of 15%.
  • the resulting autolyzed mixture was added to a compound enzyme for enzymolysis.
  • the compound enzyme contained Angel Anzhanmei’s compound enzyme preparation protease EF108.
  • the amount of protease EF108 added was 8 ⁇ based on the dry matter of the mixture.
  • the enzymolysis temperature of the compound enzyme preparation AH-1 is 55° C.
  • the pH is 6.0
  • the enzymolysis time is 12 hours.
  • the obtained protease enzymolysis mixture was added to mannanase for enzymolysis.
  • the amount of mannanase added was 8 ⁇
  • the enzymolysis temperature was 55° C.
  • the pH was 6.0
  • the enzymolysis time was 12 hours.
  • the addition amount of the ⁇ -glucanase is 6 ⁇
  • the addition amount of cellulase is 3 ⁇
  • the enzymolysis temperature is 45°C.
  • the pH is 5.0
  • the enzymatic hydrolysis time is 16 hours.
  • a three-effect or four-effect evaporation system is used to evaporate and concentrate the yeast hydrolyzate solution that has been autolyzed and enzymatically hydrolyzed, and the solid content of the concentrated product is 35-45%.
  • the concentrated paste is spray-dried, the air inlet temperature of spray drying is controlled at 155-175°C, the outlet air temperature is controlled at 90-115°C, the atomization pressure is controlled at 100-160barr, and the receiving temperature is controlled at 25-45°C .
  • the yeast species is a strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418, Saccharomyces cerevisiaed8.8, CCTCC NO:M2017148) culture solution contains carbon source, nitrogen source, phosphorus source, wherein carbon source is 30% sugarcane molasses, the nitrogen source is ammonia water with a nitrogen content of 16%, the phosphorus source is phosphoric acid, and Saccharomyces cerevisiae is inoculated for fermentation and cultivation.
  • the carbon source, nitrogen source, and phosphorus source are all added by fed-batch method, and the fermentation temperature is 30°C.
  • the fermentation time is 16 hours, the fermentation pH is controlled at 5.5, and the pure culture liquid fermented yeast milk is obtained, and 50% of the yeast milk protein is determined.
  • the yeast milk prepared by the above process of Saccharomyces cerevisiaed8.8 was used to make acidified yeast milk: based on the mass of yeast milk dry matter, 4% citric acid was added to acidify the yeast milk to obtain acidified yeast milk.
  • yeast milk prepared by the above process of Saccharomyces cerevisiae FX-2 is used to make cell wall emulsion: yeast milk autolysis treatment, the yeast autolysis process controls the salt concentration to 6%, the pH value is 6.5, and the temperature is 55°C. After autolysis for 25 hours, centrifuge After separation, a yeast cell wall emulsion was obtained with a protein content of 15%.
  • 5% cell wall emulsion is added to the acidified yeast milk for mixing to obtain a premix solution with a protein content of about 36%.
  • the mixed solution adjusts the dry matter, it is heat-shocked and put into the tank, and the temperature rises and autodissolves.
  • the dry matter content of the mixed solution is adjusted to 20% before the autolysis.
  • the autolysis temperature range is 55° C., and the holding time is 8 hours.
  • the resulting autolyzed mixture was added to a compound enzyme for enzymolysis.
  • the compound enzyme included protease EF108 from Angel Anzhanmei compound enzyme preparation.
  • the amount of protease EF108 added was 4 ⁇ based on the dry matter of the mixture.
  • the enzymolysis temperature of the compound enzyme preparation AH-1 is 60° C., the pH is 5.0, and the enzymolysis time is 6 hours.
  • the obtained protease hydrolysis mixture was added to mannanase for enzymolysis.
  • the amount of mannanase added was 4 ⁇
  • the enzymolysis temperature was 60° C.
  • the pH was 5.0
  • the enzymolysis time was 6 hours.
  • the added amount of described ⁇ -glucanase is 3 ⁇
  • the added amount of cellulase is 1 ⁇
  • the enzymatic hydrolysis temperature is 55°C.
  • the pH is 4.0
  • the enzymatic hydrolysis time is 8 hours.
  • a three-effect or four-effect evaporation system is used to evaporate and concentrate the yeast hydrolyzate solution that has been autolyzed and enzymatically hydrolyzed, and the solid content in the concentrated product is 35-45%.
  • the concentrated paste is spray-dried, the air inlet temperature of spray drying is controlled at 155-175°C, the outlet air temperature is controlled at 90-115°C, the atomization pressure is controlled at 100-160barr, and the receiving temperature is controlled at 25-45°C .
  • the yeast species is a strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418, Saccharomyces cerevisiaed8.8, CCTCC NO:M2017148) culture solution contains carbon source, nitrogen source, phosphorus source, wherein carbon source is 30% sugarcane molasses, the nitrogen source is ammonia water with a nitrogen content of 16%, the phosphorus source is phosphoric acid, and Saccharomyces cerevisiae is inoculated for fermentation and cultivation.
  • the carbon source, nitrogen source, and phosphorus source are all added by fed-batch method, and the fermentation temperature is 30°C.
  • the fermentation time is 16 hours, the fermentation pH is controlled at 5.5, and the pure culture liquid fermented yeast milk is obtained, and 50% of the yeast milk protein is determined.
  • the yeast milk prepared by the above process of Saccharomyces cerevisiaed8.8 is used to make acidified yeast milk: based on the mass of yeast milk dry matter, 1% citric acid is added to acidify the yeast milk to obtain acidified yeast milk.
  • yeast milk prepared by the above process of Saccharomyces cerevisiae FX-2 is used to make cell wall emulsion: the above-mentioned yeast milk is autolyzed, and the yeast autolysis process controls the salt concentration to 3%, the pH value is 6.5, and the temperature is 55 ° C, autolyzed for 15 hours Finally, the yeast cell wall emulsion is obtained after centrifugation, and its protein content is about 30%.
  • the resulting autolyzed mixture was added to a compound enzyme for enzymolysis.
  • the compound enzyme contained Angel Anzhanmei’s compound enzyme preparation protease EF108.
  • the amount of protease EF108 added was 8 ⁇ based on the dry matter of the mixture.
  • the enzymolysis temperature of the compound enzyme preparation AH-1 is 55° C.
  • the pH is 6.0
  • the enzymolysis time is 12 hours.
  • the obtained protease enzymolysis mixture was added to mannanase for enzymolysis.
  • the amount of mannanase added was 8 ⁇
  • the enzymolysis temperature was 55° C.
  • the pH was 6.0
  • the enzymolysis time was 12 hours.
  • the addition amount of the ⁇ -glucanase is 6 ⁇
  • the addition amount of cellulase is 3 ⁇
  • the enzymolysis temperature is 45°C.
  • the pH is 5.0
  • the enzymatic hydrolysis time is 16 hours.
  • a three-effect or four-effect evaporation system is used to evaporate and concentrate the yeast hydrolyzate solution that has been autolyzed and enzymatically hydrolyzed, and the solid content in the concentrated product is 35-45%.
  • the concentrated paste is spray-dried, the air inlet temperature of spray drying is controlled at 155-175°C, the outlet air temperature is controlled at 90-115°C, the atomization pressure is controlled at 100-160barr, and the receiving temperature is controlled at 25-45°C .
  • the yeast species is a strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418, Saccharomyces cerevisiaed8.8, CCTCC NO:M2017148) culture solution contains carbon source, nitrogen source, phosphorus source, wherein carbon source is 30% sugarcane molasses, the nitrogen source is ammonia water with a nitrogen content of 16%, and the phosphorus source is phosphoric acid. Saccharomyces cerevisiae is inoculated for fermentation and culture. The carbon source, nitrogen source, and phosphorus source are all added by fed-batch method. The fermentation time is 12 hours, the fermentation pH is controlled at 5.5, and the pure culture liquid fermented yeast milk is obtained, and 60% of the yeast milk protein is determined.
  • the yeast milk is acidified by adding 1% citric acid based on the mass of the dry matter of the yeast milk to obtain the acidified yeast milk. Yeast cell wall emulsion is not added.
  • the dry matter content of the mixed solution is adjusted to 10% before the autolysis.
  • the autolysis temperature range is 45° C., and the holding time is 14 hours.
  • the resulting autolyzed mixture was added to a compound enzyme for enzymolysis.
  • the compound enzyme contained Angel Anzhanmei’s compound enzyme preparation protease EF108.
  • the amount of protease EF108 added was 8 ⁇ based on the dry matter of the mixture.
  • the enzymolysis temperature of the compound enzyme preparation AH-1 is 55° C.
  • the pH is 6.0
  • the enzymolysis time is 12 hours.
  • the obtained protease enzymolysis mixture was added to mannanase for enzymolysis.
  • the amount of mannanase added was 8 ⁇
  • the enzymolysis temperature was 55° C.
  • the pH was 6.0
  • the enzymolysis time was 12 hours.
  • the addition amount of the ⁇ -glucanase is 6 ⁇
  • the addition amount of cellulase is 3 ⁇
  • the enzymolysis temperature is 45°C.
  • the pH is 5.0
  • the enzymatic hydrolysis time is 16 hours.
  • a three-effect or four-effect evaporation system is used to evaporate and concentrate the yeast hydrolyzate solution that has been autolyzed and enzymatically hydrolyzed, and the solid content in the concentrated product is 35-45%.
  • the concentrated paste is spray-dried, the air inlet temperature of spray drying is controlled at 155-175°C, the outlet air temperature is controlled at 90-115°C, the atomization pressure is controlled at 100-160barr, and the receiving temperature is controlled at 25-45°C .
  • the yeast species is a strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418, Saccharomyces cerevisiaed8.8, CCTCC NO:M2017148) culture solution contains carbon source, nitrogen source, phosphorus source, wherein carbon source is 30% sugarcane molasses, the nitrogen source is ammonia water with a nitrogen content of 16%, the phosphorus source is phosphoric acid, and Saccharomyces cerevisiae is inoculated for fermentation and cultivation.
  • the carbon source, nitrogen source, and phosphorus source are all added by fed-batch method, and the fermentation temperature is 30°C.
  • the fermentation time is 12 hours, the fermentation pH is controlled at 5.5, and the pure culture liquid fermented yeast milk is obtained, and 60% of the yeast milk protein is determined.
  • the yeast milk prepared by the above process of Saccharomyces cerevisiaed8.8 was used to make acidified yeast milk: based on the mass of yeast milk dry matter, 4% citric acid was added to acidify the yeast milk to obtain acidified yeast milk.
  • the yeast milk prepared by the above process of Saccharomyces cerevisiae FX-2 is used to make cell wall emulsion: yeast milk autolysis treatment, the yeast autolysis process controls the salt concentration to 3%, the pH value is 6.5, and the temperature is 55°C. After autolysis for 15 hours, centrifuge After separation, the yeast cell wall emulsion is obtained, and its protein content is about 30%.
  • the resulting autolyzed mixture was added to a compound enzyme for enzymolysis.
  • the compound enzyme included protease EF108 from Angel Anzhanmei compound enzyme preparation.
  • the amount of protease EF108 added was 4 ⁇ based on the dry matter of the mixture.
  • the enzymolysis temperature of the compound enzyme preparation AH-1 is 60° C., the pH is 5.0, and the enzymolysis time is 6 hours.
  • the obtained protease hydrolysis mixture was added to mannanase for enzymolysis.
  • the amount of mannanase added was 4 ⁇
  • the enzymolysis temperature was 60° C.
  • the pH was 5.0
  • the enzymolysis time was 6 hours.
  • the added amount of described ⁇ -glucanase is 3 ⁇
  • the added amount of cellulase is 1 ⁇
  • the enzymatic hydrolysis temperature is 55°C.
  • the pH is 4.0
  • the enzymatic hydrolysis time is 8 hours.
  • a three-effect or four-effect evaporation system is used to evaporate and concentrate the yeast hydrolyzate solution that has been autolyzed and enzymatically hydrolyzed, and the solid content in the concentrated product is 35-45%.
  • the concentrated paste is spray-dried, the air inlet temperature of spray drying is controlled at 155-175°C, the outlet air temperature is controlled at 90-115°C, the atomization pressure is controlled at 100-160barr, and the receiving temperature is controlled at 25-45°C .
  • the yeast species is a strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418, Saccharomyces cerevisiaed8.8, CCTCC NO:M2017148) culture solution contains carbon source, nitrogen source, phosphorus source, wherein carbon source is 30% sugarcane molasses, the nitrogen source is ammonia water with a nitrogen content of 16%, and the phosphorus source is phosphoric acid. Saccharomyces cerevisiae is inoculated for fermentation and culture. The carbon source, nitrogen source, and phosphorus source are all added by fed-batch method. The fermentation time is 14 hours, the fermentation pH is controlled at 5.5, and the pure culture liquid fermented yeast milk is obtained, and the yeast milk protein is determined to be 55%.
  • the yeast milk prepared by the above process of Saccharomyces cerevisiaed8.8 is used to make acidified yeast milk: based on the mass of yeast milk dry matter, 2% citric acid is added to acidify the yeast milk to obtain acidified yeast milk.
  • Yeast milk prepared by the above process of Saccharomyces cerevisiae FX-2 is used to make cell wall emulsion: the above-mentioned yeast milk is autolyzed, and the yeast autolysis process controls the salt concentration to 4%, the pH value is 6.5, and the temperature is 55 ° C, autolyzed for 20 hours Finally, the yeast cell wall emulsion is obtained after centrifugation, and its protein content is about 20%.
  • the solid content in the premix is adjusted to 15% with water, and then heat-shocked and put into the tank to heat up and autolyze.
  • the autolysis temperature range is 50°C, and the holding time is 10 hours.
  • the resulting autolyzed mixture was added to a compound enzyme for enzymolysis.
  • the compound enzyme contained Angel Anzhanmei compound enzyme preparation protease EF108, and the amount of protease EF108 added was 6 ⁇ based on the dry matter of the mixture.
  • the enzymolysis temperature of the compound enzyme preparation AH-1 is 57° C.
  • the pH is 5.5
  • the enzymolysis time is 10 hours.
  • the obtained protease hydrolysis mixture was added to mannanase for enzymolysis.
  • the amount of mannanase added was 6 ⁇
  • the enzymolysis temperature was 57° C.
  • the pH was 5.5
  • the enzymolysis time was 10 hours.
  • the addition amount of the ⁇ -glucanase is 5 ⁇
  • the addition amount of cellulase is 2 ⁇
  • the enzymolysis temperature is 50°C.
  • the pH is 4.5
  • the enzymatic hydrolysis time is 12 hours.
  • a three-effect or four-effect evaporation system is used to evaporate and concentrate the yeast hydrolyzate solution that has been autolyzed and enzymatically hydrolyzed, and the solid content in the concentrated product is 35-45%.
  • the concentrated paste is spray-dried, the air inlet temperature of spray drying is controlled at 155-175°C, the outlet air temperature is controlled at 90-115°C, the atomization pressure is controlled at 100-160barr, and the receiving temperature is controlled at 25-45°C .
  • the yeast species is a strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418, Saccharomyces cerevisiaed8.8, CCTCC NO:M2017148) culture solution contains carbon source, nitrogen source, phosphorus source, wherein carbon source is 30% sugarcane molasses, the nitrogen source is ammonia water with a nitrogen content of 16%, the phosphorus source is phosphoric acid, and Saccharomyces cerevisiae is inoculated for fermentation and cultivation.
  • the carbon source, nitrogen source, and phosphorus source are all added by fed-batch method, and the fermentation temperature is 30°C.
  • the fermentation time is 14 hours, the fermentation pH is controlled at 5.5, and the pure culture liquid fermented yeast milk is obtained, and the yeast milk protein is determined to be 55%.
  • the yeast milk prepared by the above process of Saccharomyces cerevisiaed8.8 is used to make acidified yeast milk: based on the mass of yeast milk dry matter, 3% citric acid is added to acidify the yeast milk to obtain acidified yeast milk.
  • Yeast milk prepared by the above process of Saccharomyces cerevisiae FX-2 is used to make cell wall emulsion: the above-mentioned yeast milk is autolyzed, and the yeast autolysis process controls the salt concentration to 4%, the pH value is 6.5, and the temperature is 55 ° C, autolyzed for 20 hours Finally, the yeast cell wall emulsion is obtained after centrifugation, and its protein content is about 20%.
  • the mixed solution adjusts the dry matter, it is heat-shocked and put into the tank, and the temperature rises and autodissolves.
  • the dry matter content of the mixed solution is adjusted to 15% before the autolysis.
  • the autolysis temperature range is 50° C.
  • the holding time is 10 h.
  • the resulting autolyzed mixture was added to a compound enzyme for enzymolysis.
  • the compound enzyme contained Angel Anzhanmei compound enzyme preparation protease EF108, and the amount of protease EF108 added was 6 ⁇ based on the dry matter of the mixture.
  • the enzymolysis temperature of the compound enzyme preparation AH-1 is 57° C., the pH is 5.5, and the enzymolysis time is 8 hours.
  • the obtained protease hydrolysis mixture was added to mannanase for enzymolysis.
  • the amount of mannanase added was 6 ⁇
  • the enzymolysis temperature was 57° C.
  • the pH was 5.5
  • the enzymolysis time was 8 hours.
  • the added amount of described ⁇ -glucanase is 4 ⁇
  • the added amount of cellulase is 2 ⁇
  • the enzymatic hydrolysis temperature is 50°C.
  • the pH is 4.5
  • the enzymatic hydrolysis time is 12 hours.
  • a three-effect or four-effect evaporation system is used to evaporate and concentrate the yeast hydrolyzate solution that has been autolyzed and enzymatically hydrolyzed, and the solid content in the concentrated product is 35-45%.
  • the concentrated paste is spray-dried, the air inlet temperature of spray drying is controlled at 155-175°C, the outlet air temperature is controlled at 90-115°C, the atomization pressure is controlled at 100-160barr, and the receiving temperature is controlled at 25-45°C .
  • the yeast species is a strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418, Saccharomyces cerevisiaed8.8, CCTCC NO:M2017148) culture solution contains carbon source, nitrogen source, phosphorus source, wherein carbon source is 30% sugarcane molasses, the nitrogen source is ammonia water with a nitrogen content of 16%, the phosphorus source is phosphoric acid, and Saccharomyces cerevisiae is inoculated for fermentation and cultivation.
  • the carbon source, nitrogen source, and phosphorus source are all added by fed-batch method, and the fermentation temperature is 30°C.
  • the fermentation time is 12 hours, the fermentation pH is controlled at 5.5, and the pure culture liquid fermented yeast milk is obtained, and 60% of the yeast milk protein is determined.
  • the mixed solution adjusts the dry matter, it is heat-shocked and put into the tank, and the temperature rises and autodissolves.
  • the dry matter content of the mixed solution is adjusted to 21% before the autolysis.
  • the autolysis temperature range is 60° C., and the holding time is 15 hours.
  • the resulting autolyzed mixture was added to a compound enzyme for enzymolysis.
  • the compound enzyme contained Angel Anzhanmei compound enzyme preparation protease EF108.
  • the amount of protease EF108 added was 9 ⁇ based on the dry matter of the mixture.
  • the enzymolysis temperature of the compound enzyme preparation AH-1 is 65° C.
  • the pH is 6.5
  • the enzymolysis time is 13 hours.
  • the obtained protease hydrolysis mixture was added to mannanase for enzymolysis.
  • the amount of mannanase added was 9 ⁇
  • the enzymolysis temperature was 65° C.
  • the pH was 6.5
  • the enzymolysis time was 13 hours.
  • use ⁇ -glucanase and cellulase to carry out enzymatic hydrolysis again the addition amount of described ⁇ -glucanase is 7 ⁇
  • the addition amount of cellulase is 4 ⁇
  • the enzymolysis temperature is 60°C.
  • the pH is 5.5
  • the enzymatic hydrolysis time is 17 hours.
  • a three-effect or four-effect evaporation system is used to evaporate and concentrate the yeast hydrolyzate solution that has been autolyzed and enzymatically hydrolyzed, and the solid content in the concentrated product is 35-45%.
  • the concentrated paste is spray-dried, the air inlet temperature of spray drying is controlled at 155-175°C, the outlet air temperature is controlled at 90-115°C, the atomization pressure is controlled at 100-160barr, and the receiving temperature is controlled at 25-45°C .
  • the temperature is about ⁇ 2°C
  • the pH is ⁇ 0.2
  • the mass percentage is converted to about 2%.
  • the antibacterial function detection method is as follows:
  • MH broth and MH agar medium were sterilized under high pressure at 121°C for 20 minutes, and MH agar plates were prepared for use.
  • Dissolution rate According to Q/AQJM2260-2014 Feed Raw Materials, Saccharomyces cerevisiae cell wall 5.5 implementation.
  • Fig. 1 is the bacteriostasis experiment result of yeast protein to Escherichia coli in the embodiment and comparative example
  • Fig. 2 is the bacteriostasis experiment result of yeast protein to Staphylococcus aureus in the embodiment and comparative example, wherein a corresponds to comparative example 1, b-h Corresponding to Examples 1-7 respectively.
  • the results showed that against Escherichia coli and Staphylococcus aureus, b-h had obvious inhibition zone, but a had no obvious inhibition zone against Escherichia coli and Salmonella.
  • the inhibition zones of b, c, d, f, g, and h are more complete and wider, indicating that the subsequent enzymatic activity can be carried out after mixing acidified yeast milk and yeast cell wall milk Therefore, the obtained yeast protein has better antibacterial effect.

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Abstract

本申请提供了一种具有抑菌功能的酵母蛋白及其制备方法。该方法包括：步骤S1，将酿酒酵母菌株进行液体发酵，然后分离得到酵母乳；步骤S2，将酵母乳进行酸化处理，得到酸化酵母乳；可选地，将酵母乳进行自溶、或外源酶催化水解、或机械破碎，得到细胞壁乳液；步骤S3，将酸化酵母乳和可选的细胞壁乳液混合并进行自溶，得到自溶混合液；步骤S4，采用复合蛋白酶进行第一次酶解，得到第一酶解液；步骤S5，采用甘露糖酶进行第二次酶解，得到第二酶解液；采用β-葡聚糖酶和纤维素酶进行第三次酶解，得到酵母蛋白乳液；步骤S6，将酵母蛋白乳液进行蒸发浓缩，得到具有抑菌功能的酵母蛋白。该方法能够制备得到广谱抑菌功能的酵母蛋白。

Description

具有抑菌功能的酵母蛋白及其制备方法
本申请要求申请日为2021年12月28日的中国专利申请202111631106.8的优先权。本申请引用上述中国专利申请的全文。
技术领域
本申请涉及酵母深加工领域，具体而言，涉及一种具有抑菌功能的酵母蛋白及其制备方法。
背景技术
随着我国畜牧、水产业的快速发展，对饲料的需要量越来越大，饲料蛋白源的供求矛盾日益突出，在我国蛋白原料的短缺成为必然，主要依靠进口的格局不会改变，一直以来都需要从国外大量进口高蛋白饲料原料，这样的情况给本行业一方面带来了巨大的经济压力，另一方面也给生物安全性带来了严峻挑战。
自从酵母水解物和酵母细胞壁被引入饲料原料目录以后，越来越被行业内认可，针对酵母水解物和酵母细胞壁的使用也越来越多。使用酵母水解物类酵母蛋白原料，不仅可缓解饲料蛋白质资源紧缺的矛盾，还能改善蛋白质原料的营养品质。酵母细胞壁提升免疫、促进动物机体健康的使用效果已逐步被人们接受，是一种比较理想的饲用抗生素替代品。
在现有的酵母水解物生产工艺中，能够很好在养殖领域提供营养促生长功能，然而并没有抑菌效果。随着无抗养殖时代的到来，开发具有抑菌功能的酵母蛋白产品，实现对革兰氏阴性菌(大肠杆菌)和革兰氏阳性菌(金黄色葡萄球菌)等细菌的抑制，实现广谱抑菌作用，从而实现在酵母蛋白系列产品在产品功能上的升级，具有深远的意义。在无抗养殖中，具有抑菌功能的酵母蛋白既能提供营养促进生长，又能预防细菌性疾病，在强化免疫的同时促进生长，有利于充分减少抗生素的禁用在养殖中带来的影响。
基于以上原因，提供一种制备具有抑菌功能的酵母水解物的方法，是本领域亟需解决的问题。
发明内容
本申请的主要目的在于提供一种具有抑菌功能的酵母蛋白及其制备方法，以解决现有酵母水解物及酵母细胞壁产品不具有抑菌功能，在无抗养殖过程中难以预防细菌性疾病的技术问题。
为了实现上述目的，根据本申请的一个方面，提供了一种具有抑菌功能的酵母蛋白的制备方法，其包括以下步骤：步骤S1，将酿酒酵母菌株进行液体发酵，然后分离得到酵母乳；其中酿酒酵母菌株选自酿酒酵母Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418和/或 酿酒菌株Saccharomyces cerevisiaed8.8 CCTCC NO:M2017148；步骤S2，将酵母乳进行酸化处理，得到酸化酵母乳；可选地，将酵母乳进行自溶、或外源酶催化水解、或机械破碎，得到细胞壁乳液；步骤S3，将酸化酵母乳和可选的细胞壁乳液混合并进行自溶，得到自溶混合液；其中自溶混合液的浓度以干酵母质量含量计为10～20％；步骤S4，采用复合蛋白酶对自溶混合液进行第一次酶解，得到第一酶解液；步骤S5，采用甘露糖酶对第一酶解液进行第二次酶解，得到第二酶解液；采用β-葡聚糖酶和纤维素酶对第二酶解液进行第三次酶解，得到酵母蛋白乳液；步骤S6，将酵母蛋白乳液进行蒸发浓缩，得到具有抑菌功能的酵母蛋白。
进一步地，步骤S1中，液体发酵过程中采用流加方式添加碳源、氮源和磷源，碳源为糖蜜，氮源为氨水，磷源为磷酸二氢铵溶液；优选地，糖蜜中的总糖含量为28.5～31.5％，氨水中氮元素的含量为15～17％，磷酸二氢铵溶液中的磷元素含量为9～11％；优选地，液体发酵过程中的温度为30～35℃，发酵时间为12～16h，发酵pH值为4.5～6.0；优选地，酵母乳中的蛋白含量≥50％。
进一步地，步骤S1包括：将酿酒菌株Saccharomyces cerevisiaed8.8 CCTCC NO:M2017148进行液体发酵，然后分离得到第一酵母乳；将酿酒酵母Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418进行液体发酵，然后分离得到第二酵母乳；步骤S2中，将第一酵母乳进行酸化处理，得到酸化酵母乳；将第二酵母乳进行自溶、或外源酶催化水解、或机械破碎，得到细胞壁乳液；步骤S3中，将酸化酵母乳和细胞壁乳液混合并进行自溶，得到自溶混合液。
进一步地，步骤S2中，采用柠檬酸对第一酵母乳进行酸化处理；优选地，柠檬酸的添加量为酵母乳重量的1～4％；优选地，步骤S2中，细胞壁乳液中蛋白含量为15～30％；优选地，步骤S3包括：将酸化酵母乳和细胞壁乳液混合，得到蛋白含量≥35％的预混液；将预混液调节固含量后用蒸汽升温以进行自溶，得到自溶混合液；更优选地，蒸汽升温以进行自溶的过程中，控制温度为45～55℃，保温时间为8～14h。
进一步地，复合蛋白酶包括木瓜蛋白酶、中性蛋白酶、碱性蛋白酶、酸性蛋白酶；优选地，复合蛋白酶为蛋白酶EF108。
进一步地，第一次酶解过程中，复合蛋白酶的加入量为自溶混合液中干基重量的4～8‰；优选地，第一次酶解过程的酶解温度为55～60℃，pH为5.0～6.0，酶解时间为6～12h。
进一步地，第二次酶解过程中，甘露糖酶的加入量为第一酶解液中干基重量的4～8‰；优选地，第二次酶解过程的酶解温度为55～60℃，pH为5.0～6.0，酶解时间为6～12h。
进一步地，第三次酶解过程中，β-葡聚糖酶的加入量为第二酶解液中干基重量的3～6‰，纤维素酶的加入量为第二酶解液中干基重量的1～3‰；优选地，第三次酶解过程中的酶解温度为45～55℃，pH为4.0～5.0，酶解时间为8～16h；更优选地，第三次酶解过程中，使用有机酸和/或无机酸将pH值调节至4.0～5.0；更优选地，有机酸为苹果酸、柠檬酸、乳酸中的一种或多种，无机酸为盐酸和/或硫酸，更优选采用有机酸进行调节。
进一步地，步骤S6包括：将酵母蛋白液升温至78～82℃温度下灭酶1～2h；将灭酶后的酵母蛋白液进行蒸发浓缩，直至固含量达到35～45％，得到具有抑菌功能的酵母蛋白；优选地，在蒸发浓缩步骤之后，制备方法还包括：将蒸发浓缩步骤得到的浓缩液进行喷雾干燥，得到具有抑菌功能的酵母蛋白；更优选地，喷雾干燥过程中，进风温度控制在155～175℃，出风温度控制在90～115℃，雾化压力控制在100～160bar，收料温度控制在25～45℃。
根据本申请的另一方面，还提供了一种具有抑菌功能的酵母蛋白，其由上述制备方法制备得到。
本申请采用特定的酿酒酵母菌株得到酵母乳原料与可选的酵母细胞壁原料，将酸化后的酵母乳和可选的酵母细胞壁乳液进行混合后，采用特定的酶解工艺对混合液进行酶解，后经蒸发浓缩形成了具有抑菌功能的酵母蛋白产品，其在满足酵母蛋白产品在蛋白(蛋白≥35％)及小肽等功能性指标的基础上增加抑菌功能，开发出一款具有广谱抑菌功能的酵母蛋白产品。该酵母蛋白在无抗养殖过程可以预防细菌性疾病，起到强化免疫的作用。
酿酒酵母Saccharomyces cerevisiae FX-2，于2016年8月1日保藏于中国典型微生物保藏中心(CCTCC)，保藏编号为CCTCCNO:M2016418，保藏地址：中国.武汉.武汉大学，邮政编码：430072；电话(027)-68754052。
酿酒菌株Saccharomyces cerevisiaed8.8，于2017年3月29日保藏于中国典型微生物保藏中心(CCTCC)，保藏编号为CCTCC NO:M2017148，保藏地址：中国.武汉.武汉大学，邮政编码：430072；电话(027)-68754052。
附图说明
构成本申请的一部分的说明书附图用来提供对本申请的进一步理解，本申请的示意性实施例及其说明用于解释本申请，并不构成对本申请的不当限定。在附图中：
图1示出了根据本申请实施例和对比例制备的酵母蛋白对大肠杆菌的抑菌实验结果；以及
图2示出了根据本申请实施例和对比例制备的酵母蛋白对金黄色葡萄球菌的抑菌实验结果。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本申请。
正如背景技术部分所描述的，现有酵母水解物及酵母细胞壁产品均没有对革兰氏阴性菌(大肠杆菌)和革兰氏阳性菌(金黄色葡萄球菌)的生长产生抑制作用，在抑菌圈试验中无法形成有效抑菌圈。为了解决这一问题，本申请提供了一种具有抑菌功能的酵母蛋白的制备 方法。本申请在酵母水蛋白产品上实现功能升级，实现了混合制品酵母蛋白对革兰氏阴性菌(大肠杆菌)和革兰氏阳性菌(金黄色葡萄球菌)等细菌的抑制，赋予了酵母蛋白广谱抑菌功能。
在本申请一种典型的实施方式中，提供了一种具有抑菌功能的酵母蛋白的制备方法，其包括以下步骤：步骤S1，将酿酒酵母菌株进行液体发酵，然后分离得到酵母乳；其中酿酒酵母菌株选自酿酒酵母Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418和/或酿酒菌株Saccharomyces cerevisiaed8.8 CCTCC NO:M2017148；步骤S2，将酵母乳进行酸化处理，得到酸化酵母乳；可选地，将酵母乳进行自溶、或外源酶催化水解、或机械破碎，得到细胞壁乳液；步骤S3，将酸化酵母乳和可选的细胞壁乳液混合并进行自溶，得到自溶混合液；其中自溶混合液的浓度以干酵母适量含量计为10～20％；步骤S4，采用复合蛋白酶对自溶混合液进行第一次酶解，得到第一酶解液；步骤S5，采用甘露糖酶对第一酶解液进行第二次酶解，得到第二酶解液；采用β-葡聚糖酶和纤维素酶对第二酶解液进行第三次酶解，得到酵母蛋白乳液；步骤S6，将酵母蛋白乳液进行蒸发浓缩，得到具有抑菌功能的酵母蛋白。
本申请采用特定的酿酒酵母菌株得到酵母乳原料与可选的酵母细胞壁原料(代表酵母细胞壁乳液可加可不加)，将酸化后的酵母乳和可选的酵母细胞壁乳液进行混合后，采用特定的酶解工艺对混合液进行酶解，后经蒸发浓缩形成了具有抑菌功能的酵母蛋白产品。该酵母蛋白产品中含有蛋白质、核酸、小肽、葡聚糖和甘露聚糖等，其溶解率能够达到45～65％，其在满足酵母蛋白产品在蛋白(蛋白≥35％，质量含量)及小肽等功能性指标的基础上增加抑菌功能，开发出一款具有广谱抑菌功能的酵母蛋白产品。该酵母蛋白能够代替抗生素应用于畜禽养殖领域，可以预防细菌性疾病，减少因抗生素的禁用带来的影响，起到强化免疫的作用。
为了进一步提高酵母乳原料质量，以便改善最终酵母蛋白产品性能，在一种优选的实施方式中，步骤S1中，液体发酵过程中采用流加方式添加碳源、氮源和磷源，碳源为糖蜜，氮源为氨水，磷源为磷酸二氢铵溶液(水溶液)；优选地，糖蜜中的总糖含量为28.5～31.5％(质量含量)，氨水中氮元素的含量为15～17％(质量含量)，磷酸二氢铵溶液中的磷元素含量为9～11％(质量含量)；优选地，液体发酵过程中的温度为30～35℃，发酵时间为12～16h，发酵pH值为4.5～6.0；优选地，酵母乳中的蛋白含量≥50％(质量含量)。
需说明的是，以上步骤S2中，可以将步骤S1中得到的酵母乳分为两部分，一部分用于酸化形成酸化酵母乳，另一部分用于制备细胞壁乳液，也可以分别用不同的酿酒酵母菌株制备的不同的酵母乳去制备酸化酵母乳和细胞壁乳液。当然，也可以仅采用酸化酵母乳进行后续酶解。为了进一步改善酵母蛋白抑菌性，在一种优选的实施方式中，步骤S1包括：将酿酒菌株Saccharomyces cerevisiaed8.8 CCTCC NO:M2017148进行液体发酵，然后分离得到第一酵母乳；将酿酒酵母Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418进行液体发酵，然后分离得到第二酵母乳；步骤S2中，将第一酵母乳进行酸化处理，得到酸化酵母乳；将第二酵母乳进行自溶、或外源酶催化水解、或机械破碎，得到细胞壁乳液；步骤S3中，将酸化酵母乳和所述细胞壁乳液混合并进行自溶，得到所述自溶混合液。
上述酸化的目的是为了形成酸化体系，增强内源酶活性，为了进一步加强该功效，优选地，步骤S2中，采用柠檬酸对酵母乳进行酸化处理；优选地，柠檬酸的添加量为酵母乳重量的1～4％。
在一种优选的实施方式中，步骤S2中，细胞壁乳液中蛋白含量为15～30％；优选地，步骤S3包括：将酸化酵母乳和细胞壁乳液混合，得到蛋白含量≥35％的预混液(更优选地，以固体占比计，在酸化酵母乳中添加5～35％的细胞壁乳液)；将预混液调节固含量后采用蒸汽升温以进行自溶，得到自溶混合液。将二者按照蛋白含量≥35％配液，有利于平衡最终产品中各组分的含量，同时也有利于进一步改善酵母蛋白产品的抑菌效果。通过调节、蒸汽升温(热击上罐)、自溶后，得以形成更稳定的、固含量更适宜的自溶混合液，以便后续酶解过程更稳定进行。更优选地，升温以进行自溶的过程中，控制温度为45～55℃，保温时间为8～14h。更优选地，上述调节干物质时，调整预混液中固含量为10～20％。
优选地，复合蛋白酶包括木瓜蛋白酶、中性蛋白酶、碱性蛋白酶、酸性蛋白酶；优选地，复合蛋白酶为安琪安占美复配酶制剂蛋白酶EF108。采用上述复合蛋白酶对自溶混合液进行蛋白酶酶解，酵母细胞中含有的大分子被蛋白酶解成小分子的多肽等，空间结构被打开，暴露出很多特异性及功能性基团，更有利于提高产物营养成分含量及抑菌性能。
为使蛋白酶酶解过程更为稳定，在一种优选的实施方式中，第一次酶解过程中，复合蛋白酶的加入量为自溶混合液中干基重量的4～8‰；优选地，第一次酶解过程的酶解温度为55～60℃，pH为5.0～6.0，酶解时间为6～12h。
为使甘露糖酶酶解更为稳定，在一种优选的实施方式中，第二次酶解过程中，甘露糖酶的加入量为第一酶解液中干基重量的4～8‰；优选地，第二次酶解过程的酶解温度为55～60℃，pH为5.0～6.0，酶解时间为6～12h。
为使纤维素酶酶解更为稳定，在一种优选的实施方式中，第三次酶解过程中，β-葡聚糖酶的加入量为第二酶解液中干基重量的3～6‰，纤维素酶的加入量为第二酶解液中干基重量的1～3‰；优选地，第三次酶解过程中的酶解温度为45～55℃，pH为4.0～5.0，酶解时间为8～16h。
示例性地，第三次酶解过程中，使用有机酸和/或无机酸将pH值调节至4.0～5.0；更优选地，有机酸为苹果酸、柠檬酸、乳酸中的一种或多种，无机酸为盐酸和/或硫酸，更优选采用有机酸进行调节。
在一种优选的实施方式中，步骤S6包括：将酵母蛋白液升温至78～82℃温度下灭酶1～2h；将灭酶后的酵母蛋白液进行蒸发浓缩，直至固含量达到35～45％，得到具有抑菌功能的酵母蛋白。蒸发浓缩形成膏体产品，也具有本申请所述的抑菌效果。具体蒸发浓缩过程可采用三效或者四效蒸发系统进行。
优选地，在蒸发浓缩步骤之后，制备方法还包括：将蒸发浓缩步骤得到的浓缩液进行喷雾干燥，得到具有抑菌功能的酵母蛋白。喷雾干燥后便于储存和运输。喷雾干燥的温度会直接影响产品水分及产品颜色，温度控制过低，产品颜色较浅水分含量高，难以控制产品卫生 指标，温度高，产品颜色深水分少，难以控制产品质量。更优选地，喷雾干燥过程中，进风温度控制在155～175℃，出风温度控制在90～115℃，雾化压力控制在100～160bar，收料温度(物料包装温度)控制在25～45℃。
在实际操作过程中，将酵母乳进行自溶、或外源酶催化水解、或机械破碎，得到细胞壁乳液。具体的，优选采用自溶的方法进行：将酵母乳进行自溶处理，控制盐浓度3～5％，pH值6.5，温度55℃，自溶15～25小时后，离心分离后得到酵母细胞壁沉淀物。
根据本申请的另一方面，还提供了一种具有抑菌功能的酵母蛋白，其由上述制备方法制备得到。该具有抑菌功能的酵母蛋白产品原料来源均来源于天然酵母，可以实现规模化生产。本申请所获得的具有抑菌功能的酵母蛋白，在保持粗蛋白，小肽营养成分基础上，相比单一酵母水解物增加了酵母多糖的含量，额外提升免疫功能。通过对酵母蛋白和酵母多糖的专一性酶解，在营养成分蛋白质和功能性成分小肽保持较高水平情况额外利用多糖酶解液对细菌产生抑制作用，形成了具有抑菌功能的酵母蛋白。具体地，本申请首先充分用蛋白酶对酵母细胞及细胞壁中的蛋白质进行充分酶解，使其中很多功能性基团及功能性成分暴露，然后再利用多糖酶对酵母细胞壁进行酶解，使得酵母细胞和酵母细胞壁中的抑菌活性物质充分释放，形成抑菌作用。针对目前养殖中，抗生素禁用的问题，本申请开发出具有抑菌功能的酵母蛋白能够缓解养殖中抗生物禁用带来的一系列影响，促进动物生长，提高转化率，提升养殖水平。
本申请已经通过实验小试、生产中试和大试生产，生产方法可行。
以下结合具体实施例对本申请作进一步详细描述，这些实施例不能理解为限制本申请所要求保护的范围。
实施例1
(1)酵母发酵培养
酵母菌种是酿酒酵母属菌株(酿酒酵母Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418、酿酒菌株Saccharomyces cerevisiaed8.8，CCTCC NO:M2017148)培养液含有碳源、氮源、磷源，其中碳源为30％的甘蔗糖蜜，氮源为含氮量16％的氨水，磷源为磷酸，接种酿酒酵母菌进行发酵培养，碳源、氮源、磷源均采用流加方式添加，发酵温度30℃，发酵时间17小时，发酵pH控制在5.5，获得纯培养液体发酵酵母乳，测定酵母乳蛋白质46％。
将酿酒菌株Saccharomyces cerevisiaed8.8经上述工艺制备的酵母乳用于制作酸化酵母乳：以酵母乳干物质的质量计，添加4％的柠檬酸将酵母乳进行酸化处理，得到酸化酵母乳。
将Saccharomyces cerevisiae FX-2经上述工艺制备的酵母乳用于制作细胞壁乳液：酵母乳自溶处理，酵母自溶过程控制盐浓度6％，pH值6.5，温度55℃，自溶25小时后，离心分离后得到酵母细胞壁乳液，其蛋白质含量为15％。
(2)酵母乳与酵母细胞壁混合过程
以酵母乳干物质的质量计，向酸化酵母乳中添加35％细胞壁乳液进行混合得到蛋白质含量35％左右的预混液。
(3)预混液自溶过程
用水调解预混液中的固含量至10％，然后进行热击上罐，升温自溶。其中自溶温度范围为45℃，保温时间14h。
(4)蛋白酶酶解过程
将所得的自溶混合液加入复合酶进行酶解，所述的复合酶包含安琪安占美复配酶制剂蛋白酶EF108，以混合液干物质质量计，蛋白酶EF108的添加量为8‰，所述复合酶制剂AH-1的酶解温度为55℃，pH为6.0，酶解时间为12小时。
(5)多糖酶酶解过程
将所得到的蛋白酶酶解混合液加入甘露聚糖酶进行酶解，所述甘露聚糖酶的添加量为8‰，酶解温度为55℃，pH为6.0，酶解时间12小时。然后同时利用β-葡聚糖酶和纤维素酶进行再次酶解，所述的β-葡聚糖酶的添加量为6‰，纤维素酶添加量为3‰，酶解温度为45℃，pH为5.0，酶解时间为16小时。
(6)蒸发浓缩过程
采用三效或者四效蒸发系统将自溶酶解完成的酵母水解物溶液进行蒸发浓缩，浓缩品中的固含量在35～45％。
(7)喷雾干燥
将浓缩后膏体进行喷雾干燥，喷雾干燥的进风温度控制在155～175℃，出风温度控制在90～115℃，雾化压力控制在100～160barr，收料温度控制在25～45℃。
实施例2
(1)酵母发酵培养
酵母菌种是酿酒酵母属菌株(酿酒酵母Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418、酿酒菌株d8.8Saccharomyces cerevisiaed8.8，CCTCC NO:M2017148)培养液含有碳源、氮源、磷源，其中碳源为30％的甘蔗糖蜜，氮源为含氮量16％的氨水，磷源为磷酸，接种酿酒酵母菌进行发酵培养，碳源、氮源、磷源均采用流加方式添加，发酵温度30℃，发酵时间16小时，发酵pH控制在5.5，获得纯培养液体发酵酵母乳，测定酵母乳蛋白质50％。
将酿酒菌株Saccharomyces cerevisiaed8.8经上述工艺制备的酵母乳用于制作酸化酵母乳：以酵母乳干物质的质量计，添加4％的柠檬酸将酵母乳进行酸化处理，得到酸化酵母乳。
将Saccharomyces cerevisiae FX-2经上述工艺制备的酵母乳用于制作细胞壁乳液：酵母乳自溶处理，酵母自溶过程控制盐浓度6％，pH值6.5，温度55℃，自溶25小时后，离心分离后得到酵母细胞壁乳液，其蛋白质含量为15％。
(2)酵母乳与酵母细胞壁混合过程
以酵母乳干物质的质量计，向酸化酵母乳中添加5％细胞壁乳液进行混合得到蛋白质含量36％左右的预混液。
(3)混合液自溶过程
混合液调解干物质后进行热击上罐，升温自溶。其中，所述自溶前调节混合液干物质含量20％。其中，所述的自溶温度范围为55℃，保温时间8h。
(4)蛋白酶酶解过程
将所得的自溶混合液加入复合酶进行酶解，所述的复合酶包含安琪安占美复配酶制剂蛋白酶EF108，以混合液干物质质量计，蛋白酶EF108的添加量为4‰，所述复合酶制剂AH-1的酶解温度为60℃，pH为5.0，酶解时间为6小时。
(5)多糖酶酶解过程
将所得到的蛋白酶酶解混合液加入甘露聚糖酶进行酶解，所述甘露聚糖酶的添加量为4‰，酶解温度为60℃，pH为5.0，酶解时间6小时。然后同时利用β-葡聚糖酶和纤维素酶进行再次酶解，所述的β-葡聚糖酶的添加量为3‰，纤维素酶添加量为1‰，酶解温度为55℃，pH为4.0，酶解时间为8小时。
(6)蒸发浓缩过程
采用三效或者四效蒸发系统将自溶酶解完成的酵母水解物溶液进行蒸发浓缩，浓缩品中的固形物含量在35～45％。
(7)喷雾干燥
将浓缩后膏体进行喷雾干燥，喷雾干燥的进风温度控制在155～175℃，出风温度控制在90～115℃，雾化压力控制在100～160barr，收料温度控制在25～45℃。
实施例3
(1)酵母发酵培养
酵母菌种是酿酒酵母属菌株(酿酒酵母Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418、酿酒菌株d8.8Saccharomyces cerevisiaed8.8，CCTCC NO:M2017148)培养液含有碳源、氮源、磷源，其中碳源为30％的甘蔗糖蜜，氮源为含氮量16％的氨水，磷源为磷酸，接种酿酒酵母菌进行发酵培养，碳源、氮源、磷源均采用流加方式添加，发酵温度30℃，发酵时间16小时，发酵pH控制在5.5，获得纯培养液体发酵酵母乳，测定酵母乳蛋白质50％。
将酿酒菌株Saccharomyces cerevisiaed8.8经上述工艺制备的酵母乳用于制作酸化酵母乳：以酵母乳干物质的质量计，添加1％的柠檬酸将酵母乳进行酸化处理，得到酸化酵母乳。
将Saccharomyces cerevisiae FX-2经上述工艺制备的酵母乳用于制作细胞壁乳液：将上述的酵母乳自溶处理，酵母自溶过程控制盐浓度3％，pH值6.5，温度55℃，自溶15小时后，离心分离后得到酵母细胞壁乳液，其蛋白质含量为30％左右。
(2)酵母乳与酵母细胞壁混合过程
以酵母乳干物质的质量计，向酸化酵母乳中添加30％细胞壁乳液进行混合得到蛋白质含量为45％左右的预混液。
(3)预混液自溶过程
用水调解预混液中的固含量至10％，然后进行热击上罐，升温自溶。其中自溶温度范围为45℃，保温时间14h。
(4)蛋白酶酶解过程
将所得的自溶混合液加入复合酶进行酶解，所述的复合酶包含安琪安占美复配酶制剂蛋白酶EF108，以混合液干物质质量计，蛋白酶EF108的添加量为8‰，所述复合酶制剂AH-1的酶解温度为55℃，pH为6.0，酶解时间为12小时。
(5)多糖酶酶解过程
将所得到的蛋白酶酶解混合液加入甘露聚糖酶进行酶解，所述甘露聚糖酶的添加量为8‰，酶解温度为55℃，pH为6.0，酶解时间12小时。然后同时利用β-葡聚糖酶和纤维素酶进行再次酶解，所述的β-葡聚糖酶的添加量为6‰，纤维素酶添加量为3‰，酶解温度为45℃，pH为5.0，酶解时间为16小时。
(6)蒸发浓缩过程
采用三效或者四效蒸发系统将自溶酶解完成的酵母水解物溶液进行蒸发浓缩，浓缩品中的固形物含量在35～45％。
(7)喷雾干燥
将浓缩后膏体进行喷雾干燥，喷雾干燥的进风温度控制在155～175℃，出风温度控制在90～115℃，雾化压力控制在100～160barr，收料温度控制在25～45℃。
实施例4
(1)酵母发酵培养
酵母菌种是酿酒酵母属菌株(酿酒酵母Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418、酿酒菌株d8.8Saccharomyces cerevisiaed8.8，CCTCC NO:M2017148)培养液含有碳源、氮源、磷源，其中碳源为30％的甘蔗糖蜜，氮源为含氮量16％的氨水，磷源为磷酸， 接种酿酒酵母菌进行发酵培养，碳源、氮源、磷源均采用流加方式添加，发酵温度30℃，发酵时间12小时，发酵pH控制在5.5，获得纯培养液体发酵酵母乳，测定酵母乳蛋白质60％。
(2)酵母乳酸化
以酵母乳干物质的质量计，添加1％的柠檬酸将酵母乳进行酸化处理，得到酸化酵母乳。不添加酵母细胞壁乳液。
(3)自溶过程
酸化酵母乳调解干物质后进行热击上罐，升温自溶。其中，所述自溶前调节混合液干物质含量10％。其中，所述的自溶温度范围为45℃，保温时间14h。
(4)蛋白酶酶解过程
将所得的自溶混合液加入复合酶进行酶解，所述的复合酶包含安琪安占美复配酶制剂蛋白酶EF108，以混合液干物质质量计，蛋白酶EF108的添加量为8‰，所述复合酶制剂AH-1的酶解温度为55℃，pH为6.0，酶解时间为12小时。
(5)多糖酶酶解过程
将所得到的蛋白酶酶解混合液加入甘露聚糖酶进行酶解，所述甘露聚糖酶的添加量为8‰，酶解温度为55℃，pH为6.0，酶解时间12小时。然后同时利用β-葡聚糖酶和纤维素酶进行再次酶解，所述的β-葡聚糖酶的添加量为6‰，纤维素酶添加量为3‰，酶解温度为45℃，pH为5.0，酶解时间为16小时。
(6)蒸发浓缩过程
采用三效或者四效蒸发系统将自溶酶解完成的酵母水解物溶液进行蒸发浓缩，浓缩品中的固形物含量在35～45％。
(7)喷雾干燥
将浓缩后膏体进行喷雾干燥，喷雾干燥的进风温度控制在155～175℃，出风温度控制在90～115℃，雾化压力控制在100～160barr，收料温度控制在25～45℃。
实施例5
(1)酵母发酵培养
酵母菌种是酿酒酵母属菌株(酿酒酵母Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418、酿酒菌株d8.8Saccharomyces cerevisiaed8.8，CCTCC NO:M2017148)培养液含有碳源、氮源、磷源，其中碳源为30％的甘蔗糖蜜，氮源为含氮量16％的氨水，磷源为磷酸，接种酿酒酵母菌进行发酵培养，碳源、氮源、磷源均采用流加方式添加，发酵温度30℃，发酵时间12小时，发酵pH控制在5.5，获得纯培养液体发酵酵母乳，测定酵母乳蛋白质60％。
将酿酒菌株Saccharomyces cerevisiaed8.8经上述工艺制备的酵母乳用于制作酸化酵母乳：以酵母乳干物质的质量计，添加4％的柠檬酸将酵母乳进行酸化处理，得到酸化酵母乳。
将Saccharomyces cerevisiae FX-2经上述工艺制备的酵母乳用于制作细胞壁乳液：酵母乳自溶处理，酵母自溶过程控制盐浓度3％，pH值6.5，温度55℃，自溶15小时后，离心分离后得到酵母细胞壁乳液，其蛋白质含量为30％左右。
(2)酵母乳与酵母细胞壁混合过程
以酵母乳干物质的质量计，向酸化酵母乳中添加30％细胞壁乳液进行混合得到蛋白质含量为50％左右的预混液。
(3)混合液自溶过程
用水调解预混液中的固含量至20％，然后进行热击上罐，升温自溶。其中自溶温度范围为55℃，保温时间8h。
(4)蛋白酶酶解过程
将所得的自溶混合液加入复合酶进行酶解，所述的复合酶包含安琪安占美复配酶制剂蛋白酶EF108，以混合液干物质质量计，蛋白酶EF108的添加量为4‰，所述复合酶制剂AH-1的酶解温度为60℃，pH为5.0，酶解时间为6小时。
(5)多糖酶酶解过程
将所得到的蛋白酶酶解混合液加入甘露聚糖酶进行酶解，所述甘露聚糖酶的添加量为4‰，酶解温度为60℃，pH为5.0，酶解时间6小时。然后同时利用β-葡聚糖酶和纤维素酶进行再次酶解，所述的β-葡聚糖酶的添加量为3‰，纤维素酶添加量为1‰，酶解温度为55℃，pH为4.0，酶解时间为8小时。
(6)蒸发浓缩过程
采用三效或者四效蒸发系统将自溶酶解完成的酵母水解物溶液进行蒸发浓缩，浓缩品中的固形物含量在35～45％。
(7)喷雾干燥
将浓缩后膏体进行喷雾干燥，喷雾干燥的进风温度控制在155～175℃，出风温度控制在90～115℃，雾化压力控制在100～160barr，收料温度控制在25～45℃。
实施例6
(1)酵母发酵培养
酵母菌种是酿酒酵母属菌株(酿酒酵母Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418、酿酒菌株d8.8Saccharomyces cerevisiaed8.8，CCTCC NO:M2017148)培养液含有碳源、氮源、磷源，其中碳源为30％的甘蔗糖蜜，氮源为含氮量16％的氨水，磷源为磷酸， 接种酿酒酵母菌进行发酵培养，碳源、氮源、磷源均采用流加方式添加，发酵温度30℃，发酵时间14小时，发酵pH控制在5.5，获得纯培养液体发酵酵母乳，测定酵母乳蛋白质55％。
将酿酒菌株Saccharomyces cerevisiaed8.8经上述工艺制备的酵母乳用于制作酸化酵母乳：以酵母乳干物质的质量计，添加2％的柠檬酸将酵母乳进行酸化处理，得到酸化酵母乳。
将Saccharomyces cerevisiae FX-2经上述工艺制备的酵母乳用于制作细胞壁乳液：将上述的酵母乳自溶处理，酵母自溶过程控制盐浓度4％，pH值6.5，温度55℃，自溶20小时后，离心分离后得到酵母细胞壁乳液，其蛋白质含量为20％左右。
(2)酵母乳与酵母细胞壁混合过程
以酵母乳干物质的质量计，向酸化酵母乳中添加20％细胞壁乳液进行混合得到蛋白质含量为48％左右的预混液。
(3)预混液自溶过程
用水调解预混液中的固含量至15％，然后进行热击上罐，升温自溶。其中自溶温度范围为50℃，保温时间10h。
(4)蛋白酶酶解过程
将所得的自溶混合液加入复合酶进行酶解，所述的复合酶包含安琪安占美复配酶制剂蛋白酶EF108，以混合液干物质质量计，蛋白酶EF108的添加量为6‰，所述复合酶制剂AH-1的酶解温度为57℃，pH为5.5，酶解时间为10小时。
(5)多糖酶酶解过程
将所得到的蛋白酶酶解混合液加入甘露聚糖酶进行酶解，所述甘露聚糖酶的添加量为6‰，酶解温度为57℃，pH为5.5，酶解时间10小时。然后同时利用β-葡聚糖酶和纤维素酶进行再次酶解，所述的β-葡聚糖酶的添加量为5‰，纤维素酶添加量为2‰，酶解温度为50℃，pH为4.5，酶解时间为12小时。
(6)蒸发浓缩过程
采用三效或者四效蒸发系统将自溶酶解完成的酵母水解物溶液进行蒸发浓缩，浓缩品中的固形物含量在35～45％。
(7)喷雾干燥
将浓缩后膏体进行喷雾干燥，喷雾干燥的进风温度控制在155～175℃，出风温度控制在90～115℃，雾化压力控制在100～160barr，收料温度控制在25～45℃。
实施例7
(1)酵母发酵培养
酵母菌种是酿酒酵母属菌株(酿酒酵母Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418、酿酒菌株d8.8Saccharomyces cerevisiaed8.8，CCTCC NO:M2017148)培养液含有碳源、氮源、磷源，其中碳源为30％的甘蔗糖蜜，氮源为含氮量16％的氨水，磷源为磷酸，接种酿酒酵母菌进行发酵培养，碳源、氮源、磷源均采用流加方式添加，发酵温度30℃，发酵时间14小时，发酵pH控制在5.5，获得纯培养液体发酵酵母乳，测定酵母乳蛋白质55％。
将酿酒菌株Saccharomyces cerevisiaed8.8经上述工艺制备的酵母乳用于制作酸化酵母乳：以酵母乳干物质的质量计，添加3％的柠檬酸将酵母乳进行酸化处理，得到酸化酵母乳。
将Saccharomyces cerevisiae FX-2经上述工艺制备的酵母乳用于制作细胞壁乳液：将上述的酵母乳自溶处理，酵母自溶过程控制盐浓度4％，pH值6.5，温度55℃，自溶20小时后，离心分离后得到酵母细胞壁乳液，其蛋白质含量为20％左右。
(2)酵母乳与酵母细胞壁混合过程
以酵母乳干物质的质量计，向酸化酵母乳中添加10％细胞壁乳液进行混合得到蛋白质含量为50％左右的预混液。
(3)混合液自溶过程
混合液调解干物质后进行热击上罐，升温自溶。其中，所述自溶前调节混合液干物质含量15％。其中，所述的自溶温度范围为50℃，保温时间10h。
(4)蛋白酶酶解过程
将所得的自溶混合液加入复合酶进行酶解，所述的复合酶包含安琪安占美复配酶制剂蛋白酶EF108，以混合液干物质质量计，蛋白酶EF108的添加量为6‰，所述复合酶制剂AH-1的酶解温度为57℃，pH为5.5，酶解时间为8小时。
(5)多糖酶酶解过程
将所得到的蛋白酶酶解混合液加入甘露聚糖酶进行酶解，所述甘露聚糖酶的添加量为6‰，酶解温度为57℃，pH为5.5，酶解时间8小时。然后同时利用β-葡聚糖酶和纤维素酶进行再次酶解，所述的β-葡聚糖酶的添加量为4‰，纤维素酶添加量为2‰，酶解温度为50℃，pH为4.5，酶解时间为12小时。
(6)蒸发浓缩过程
采用三效或者四效蒸发系统将自溶酶解完成的酵母水解物溶液进行蒸发浓缩，浓缩品中的固形物含量在35～45％。
(7)喷雾干燥
将浓缩后膏体进行喷雾干燥，喷雾干燥的进风温度控制在155～175℃，出风温度控制在90～115℃，雾化压力控制在100～160barr，收料温度控制在25～45℃。
对比例1
(1)酵母发酵培养
酵母菌种是酿酒酵母属菌株(酿酒酵母Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418、酿酒菌株d8.8Saccharomyces cerevisiaed8.8，CCTCC NO:M2017148)培养液含有碳源、氮源、磷源，其中碳源为30％的甘蔗糖蜜，氮源为含氮量16％的氨水，磷源为磷酸，接种酿酒酵母菌进行发酵培养，碳源、氮源、磷源均采用流加方式添加，发酵温度30℃，发酵时间12小时，发酵pH控制在5.5，获得纯培养液体发酵酵母乳，测定酵母乳蛋白质60％。
(2)酵母乳与酵母细胞壁混合过程
不进行酵母乳酸化，不添加细胞壁乳。
(3)混合液自溶过程
混合液调解干物质后进行热击上罐，升温自溶。其中，所述自溶前调节混合液干物质含量21％。其中，所述的自溶温度范围为60℃，保温时间15h。
(4)蛋白酶酶解过程
将所得的自溶混合液加入复合酶进行酶解，所述的复合酶包含安琪安占美复配酶制剂蛋白酶EF108，以混合液干物质质量计，蛋白酶EF108的添加量为9‰，所述复合酶制剂AH-1的酶解温度为65℃，pH为6.5，酶解时间为13小时。
(5)多糖酶酶解过程
将所得到的蛋白酶酶解混合液加入甘露聚糖酶进行酶解，所述甘露聚糖酶的添加量为9‰，酶解温度为65℃，pH为6.5，酶解时间13小时。然后同时利用β-葡聚糖酶和纤维素酶进行再次酶解，所述的β-葡聚糖酶的添加量为7‰，纤维素酶添加量为4‰，酶解温度为60℃，pH为5.5，酶解时间为17小时。
(6)蒸发浓缩过程
采用三效或者四效蒸发系统将自溶酶解完成的酵母水解物溶液进行蒸发浓缩，浓缩品中的固形物含量在35～45％。
(7)喷雾干燥
将浓缩后膏体进行喷雾干燥，喷雾干燥的进风温度控制在155～175℃，出风温度控制在90～115℃，雾化压力控制在100～160barr，收料温度控制在25～45℃。
补充说明，以上实施过程中，在端点值的取舍中，由于设备及人工操作的误差性存在，均存在一定的波动范围，温度±2℃左右，pH±0.2，质量百分比折算2％左右。
抑菌功能检测方法如下：
1、培养基的准备。
将MH肉汤、MH琼脂培养基121℃，高压灭菌20min中，并制作MH琼脂平板备用。
2、菌种培养
将保存好的大肠杆菌、金黄色葡萄球菌接种于MH肉汤，37℃，恒温摇床孵育12-14h。将复苏后的大肠杆菌和金黄色葡萄球菌重新接种MH肉汤，培养16-20h左右。经分光光度计检测，OD值在04-0.7之间。活菌数约为10 10CFU/mL
3、抑菌圈试验
3.1含菌平皿的制作
用生理盐水(经121℃，高压灭菌20min)，将步骤2.2中的纯培养菌液稀释1000倍，制作菌悬液；
将灭菌后的MH琼脂倾倒平皿，等MH平皿凝固后，每个平皿滴加200μL菌悬液。
用灭菌后的玻璃棒，快速将菌液均匀涂布整个平皿，制作含菌平皿。
3.2液体样品蒸发浓缩至40％的干物质浓度加入放在含菌平皿牛津杯(每个加250μL左右)，使液面与牛津杯相平，固体样品直接采用药品勺挑取适量约1～3g左右放入含菌平皿中。
3.3将3.2中的平皿置于恒温培养箱，37℃培养14h左右。
其他指标测试方法：
蛋白质含量：采用GB/T 6432-2018测试；
溶解率：按照Q/AQJM2260-2014饲料原料，酿酒酵母细胞壁中5.5执行。
指标分析结果见表1：
表1
Figure PCTCN2022142484-appb-000001
抑菌圈图：
图1为实施例和对比例中酵母蛋白对大肠杆菌的抑菌实验结果，图2为实施例和对比例中酵母蛋白对金黄色葡萄球菌的抑菌实验结果，其中a对应对比例1，b-h分别对应实施例1-7。结果显示，针对大肠杆菌和金黄色葡萄球菌，b-h均有明显的抑菌圈，而a针对大肠杆菌和沙门氏菌抑菌圈均不明显。尤其是，相比于e(实施例4)，b、c、d、f、g、h的抑菌圈更完整，且宽度较宽，表明将酸化酵母乳和酵母细胞壁乳混合后进行后续酶解，得到的酵母蛋白具有更好的抑菌效果。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。

Claims (10)

  1. 一种具有抑菌功能的酵母蛋白的制备方法，其特征在于，包括以下步骤：
    步骤S1，将酿酒酵母菌株进行液体发酵，然后分离得到酵母乳；其中所述酿酒酵母菌株选自酿酒酵母Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418和/或酿酒菌株Saccharomyces cerevisiaed8.8 CCTCC NO:M2017148；
    步骤S2，将所述酵母乳进行酸化处理，得到酸化酵母乳；可选地，将所述酵母乳进行自溶、或外源酶催化水解、或机械破碎，得到细胞壁乳液；
    步骤S3，将所述酸化酵母乳和可选的所述细胞壁乳液混合并进行自溶，得到自溶混合液；其中所述自溶混合液的浓度以干酵母质量含量计为10～20％；
    步骤S4，采用复合蛋白酶对所述自溶混合液进行第一次酶解，得到第一酶解液；
    步骤S5，采用甘露糖酶对所述第一酶解液进行第二次酶解，得到第二酶解液；采用β-葡聚糖酶和纤维素酶对所述第二酶解液进行第三次酶解，得到酵母蛋白乳液；
    步骤S6，将所述酵母蛋白乳液进行蒸发浓缩，得到所述具有抑菌功能的酵母蛋白。
  2. 根据权利要求1所述的制备方法，其特征在于，所述步骤S1中，所述液体发酵过程中采用流加方式添加碳源、氮源和磷源，所述碳源为糖蜜，所述氮源为氨水，所述磷源为磷酸二氢铵溶液；
    优选地，所述糖蜜中的总糖含量为28.5～31.5％，所述氨水中氮元素的含量为15～17％，所述磷酸二氢铵溶液中的磷元素含量为9～11％；
    优选地，所述液体发酵过程中的温度为30～35℃，发酵时间为12～16h，发酵pH值为4.5～6.0；
    优选地，所述酵母乳中的蛋白含量≥50％。
  3. 根据权利要求1或2所述的制备方法，其特征在于，所述步骤S1包括：将所述酿酒菌株Saccharomyces cerevisiaed8.8 CCTCC NO:M2017148进行所述液体发酵，然后分离得到第一酵母乳；将所述酿酒酵母Saccharomyces cerevisiae FX-2 CCTCCNO:M2016418进行所述液体发酵，然后分离得到第二酵母乳；所述步骤S2中，将所述第一酵母乳进行所述酸化处理，得到所述酸化酵母乳；将所述第二酵母乳进行所述自溶、或所述外源酶催化水解、或所述机械破碎，得到所述细胞壁乳液；所述步骤S3中，将所述酸化酵母乳和所述细胞壁乳液混合并进行自溶，得到所述自溶混合液。
  4. 根据权利要求1至3中任一项所述的制备方法，其特征在于，所述步骤S2中，采用柠檬酸对所述第一酵母乳进行所述酸化处理；优选地，所述柠檬酸的添加量为所述酵母乳重量的1～4％；优选地，所述步骤S2中，所述细胞壁乳液中蛋白含量为15～30％；
    优选地，所述步骤S3包括：将所述酸化酵母乳和所述细胞壁乳液混合，得到蛋白含量≥35％的预混液；将所述预混液调节固含量后用蒸汽升温以进行自溶，得到所述自溶 混合液；
    更优选地，所述蒸汽升温以进行自溶的过程中，控制温度为45～55℃，保温时间为8～14h。
  5. 根据权利要求1至4中任一项所述的制备方法，其特征在于，所述复合蛋白酶包括木瓜蛋白酶、中性蛋白酶、碱性蛋白酶、酸性蛋白酶；优选地，所述复合蛋白酶为蛋白酶EF108。
  6. 根据权利要求6所述的制备方法，其特征在于，所述第一次酶解过程中，所述复合蛋白酶的加入量为所述自溶混合液中干基重量的4～8‰；优选地，所述第一次酶解过程的酶解温度为55～60℃，pH为5.0～6.0，酶解时间为6～12h。
  7. 根据权利要求1至6中任一项所述的制备方法，其特征在于，所述第二次酶解过程中，所述甘露糖酶的加入量为所述第一酶解液中干基重量的4～8‰；优选地，所述第二次酶解过程的酶解温度为55～60℃，pH为5.0～6.0，酶解时间为6～12h。
  8. 根据权利要求7所述的制备方法，其特征在于，所述第三次酶解过程中，所述β-葡聚糖酶的加入量为所述第二酶解液中干基重量的3～6‰，所述纤维素酶的加入量为所述第二酶解液中干基重量的1～3‰；优选地，所述第三次酶解过程中的酶解温度为45～55℃，pH为4.0～5.0，酶解时间为8～16h；
    更优选地，所述第三次酶解过程中，使用有机酸和/或无机酸将pH值调节至4.0～5.0；
    更优选地，所述有机酸为苹果酸、柠檬酸、乳酸中的一种或多种，所述无机酸为盐酸和/或硫酸，更优选采用所述有机酸进行调节。
  9. 根据权利要求1至8中任一项所述的制备方法，其特征在于，所述步骤S6包括：
    将所述酵母蛋白液升温至78～82℃温度下灭酶1～2h；
    将灭酶后的所述酵母蛋白液进行蒸发浓缩，直至固含量达到35～45％，得到所述具有抑菌功能的酵母蛋白；
    优选地，在所述蒸发浓缩步骤之后，所述制备方法还包括：将所述蒸发浓缩步骤得到的浓缩液进行喷雾干燥，得到所述具有抑菌功能的酵母蛋白；
    更优选地，所述喷雾干燥过程中，进风温度控制在155～175℃，出风温度控制在90～115℃，雾化压力控制在100～160bar，收料温度控制在25～45℃。
  10. 一种具有抑菌功能的酵母蛋白，其特征在于，由权利要求1至9中任一项所述的制备方法制备得到。
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